| Assay Method Information | |
| | PANK Activity Assay |
| Description: | PANK activity assay was performed in the presence of 0-10 μM compound in a reaction mixture that contained 100 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 2.5 mM ATP, 45 μM D-[1-14C]pantothenate (specific activity, 22.5 mCi/mmol) and 5 nM of human PANK3. PANK3 concentrations were calculated using the extinction coefficient at 280 nm of 39,225M-1 cm−1. The assay was linear with time and after 10 min at 37° C. the reaction was stopped by the addition of 4 μl of 10% (v/v) acetic acid. The mixture was spotted onto a DE81 disk, washed with three successive changes of 1% acetic acid in 95% ethanol and product formation determined by scintillation counting of the dried disc. If the IC50 was determined to be in the nM range then the assay was repeated in the presence of 0-1 μM or 0-0.1 μM compound to more precisely determine the IC50. All the experiments were repeated twice in duplicate and the data were an average ±data range. For the kinetic experiments, the assay was done either varying the pantothenate from 0-180 μM or ATP from 0-125 μM at a given concentration of test compound. The experiments mimicking the mixture of ligands present in vivo were performed under different conditions. The reaction mix for the determination of the acetyl-CoA IC51 contained 100 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM ATP, 45 μM D-[1-14C]pantothenate (specific activity 22.5 mCi/mmol), 2.5 μM test compound and 1 μg of PANK3. In the time-course experiments, the reaction mixtures contained 100 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM ATP, 45 μM or 90 μM D-[1-14C]pantothenate (specific activity, 22.5 mCi/mmol), 100 μM acetyl CoA±2.5 μM test compound, and 1 μg of PANK3. |
| Affinity data for this assay | |
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